For comparison with chemically fixed osteoclasts, we prepared chick osteoclasts by quick freezing followed by freeze-substitution. In spite of technical difficulties this demonstrated that osteoclasts can be satisfactorily frozen in situ by the metal contact method. Ultrastructural differences were revealed between conventional fixation and quick freezing. Compared with conventional fixation, the quick freezing method appeared to improve preservation: (1) a discrete trilaminar plasma membrane and other intracellular membranes showed a smooth profile without undulation or rupture; (2) cytoskeletal components appeared to be clearer, straighter, and more numerous; (3) the interior of the ruffled finger contained interconnected lattice structures whereas highly organised microfilaments were seen in the clear zone; (4) well developed tubulovesicular structures (TVSs) that branched or anastomosed with each other were revealed in the cytoplasm; (5) the contents of intracellular membrane systems including the nuclear envelope, endoplasmic reticulum, and Golgi complex were stained to a various extent; (6) vesicles and vacuoles were much smaller, round and well-defined with electron-dense contents; (7) crystalline structures were seen at the extracellular channels of the ruffled border, in the lumen of TVSs, and in vesicles; (8) in some instances mitochondrial granules were visible; (9) within the resorptive lacuna, osteoclasts adhered to the degraded bone matrix without any intervening empty space.

A correspondence item entitled ‘A warning against revival of the classic tenets of gross anatomy related to nerve–muscle specificity’ was recently published in the Journal of Anatomy (Shinohara, 1996a). The present correspondent fully agrees with some aspects of his opinion, in particular, the 3 laws about applications which he described, and he also appreciates his approach to the argument based on the results of embryology and experimentation. However, his denial of morphological interpretations based on the theory of nerve–muscle specificity may be due to an incomplete understanding of the current status of the theory.

The structure of porcine skin as examined by light microscopy is reviewed and its similarities to and differences from human skin are highlighted. Special imaging techniques and staining procedures are described and their use in gathering morphological information in porcine skin is discussed. Confocal laser scanning microscopy (CLSM) was employed to examine the structure of porcine skin and the findings are presented as an adjunct to the information already available in the literature. It is concluded that CLSM provides valuable additional morphological information to material examined by conventional microscopy and is useful for wound healing studies in the porcine model.

In this paper we examine metamerism in the vertebral column of certain mammals from the perspectives of development and adaptation. To this end we examine the patterns of metameric variation of dimensions of the neural (vertebral) canal, vertebral body and spinous process in man and inbred strains of mice. The data from inbred strains of mice indicate that variability in dimensions within a strain reflects the temporal ordering and nature of developmental influences on vertebral morphogenesis. Differences between strains parallel the within-strain findings. These findings are attributed to somatic and neural influences on morphogenesis. Comparisons between mice and man indicate that these same influences can be invoked to explain and interpret the mosaic nature of vertebral column evolution. These findings lead us to conclude that different vertebral elements and levels are subject to different interactions of evolutionary and morphogenetic influences. The study of these influences and their interactions should prove fruitful in developing an understanding of the relationship between adaptation, development, growth and function in the skeleton generally.

Four forelimbs of 3 platypuses and 3 forelimbs of 2 echidnas were examined to study the precise form of the brachial plexus and to clarify the structural characteristics of the brachial plexus in phylogeny. The spinal components contributing to the plexus (C4–T2) and the formation patterns of the 3 trunks of the plexus were the same as those generally observed in mammals. In the cranial half of the brachial plexus from C4, 5 and 6 in monotremes, division into the ventral bundle (lateral cord) and dorsal bundle (axillary nerve) is clear, as in other mammals. However, for monotremes, in the caudal half of the plexus from C7 and T1 (+T2) and the nerves arising from the caudal plexus there is no definite division into the ventral and dorsal bundles, which distribute to the flexor and extensor parts of the forelimbs, respectively. The lower trunk of the monotreme brachial plexus forms a cord which contains both ventral and dorsal components. This characteristic diverges from the generally accepted idea that the tetrapod limb plexus is divided clearly into 2 layers: a dorsal layer for extensors and a ventral layer for flexors of the limb. Considering the incomplete dorsoventral division of forelimb nerves in some reptiles and urodeles, the caudal half of the monotreme brachial plexus has characteristics in common with those of lower tetrapods.

A symposium on extramedullary haematopoiesis was held on 11–12 September 1996 at the University of St Andrews. The following are abstracts of papers and posters presented at the meeting.

The relationship between IGF-I and changes in muscle fibre phenotype in response to 6 d of stretch or disuse of the lower limb muscles of the rabbit was studied by combining in situ hybridisation and immunohistochemistry procedures. Passive stretch by plaster cast immobilisation of the muscle in its lengthened position not only induced an increase in IGF-I mRNA expression within the individual muscle fibres but also an increase in the percentage of fibres expressing neonatal and slow myosin. This change in phenotype was also found to be accompanied by a rapid and marked increase of muscle mass, total RNA content as well as IGF-I gene expression. In contrast, IGF-I appears not to be involved in muscle atrophy induced by immobilisation in the shortened position and the inactivity which results from this procedure. The level of increase in expression of IGF-I mRNA varied from fibre to fibre. By using adjacent serial sections, the fibres which expressed IGF-I mRNA at the highest levels were identified as expressing neonatal and the slow type 1 myosin. These data suggest that the expression of IGF-I within individual muscle fibres is correlated not only with hypertrophy but also with the muscle phenotypic adaptation that results from stretch and overload.

Morphometric analyses of the neurons and microvessels of perfusion-fixed hypogastric (HG) and 13th thoracic (T13) ganglia have been performed in male Wistar rats aged 4, 24 and 30 mo. Estimations of HG volume employing the Cavalieri principle have also been performed and showed that the size of the aged HG is increased by 42%. Routine histological staining of the ganglia with Masson's trichrome indicated that this may be due to the increased amount of interstitial connective tissue which was apparent in the aged animals. The number of neurons per unit area progressively decreased by 38% between ages 4 and 24 mo and by 16% between ages 24 and 30 mo in the HG and by 25% (4 and 24 mo) and 2% (24 and 30 mo) in the T13 ganglion. The total number of neurons in the HG however, estimated by a physical disector analysis, was constant with age. The number of microvessels per unit area, microvessel diameter, neuronal and nuclear areas did not differ significantly between the 3 age groups studied. This observed increase in ganglionic volume and decrease in neuronal packing density may be associated with changes in the extracellular matrix, in particular in glycosaminoglycans whose presence was indicated by metachromasia of the ganglia with toluidine blue. The extracellular matrix was therefore characterised using a panel of monoclonal antibodies against glycosaminoglycans and laminin. Chondroitin-6 sulphate and chondroitin-4 sulphate were present in the interstitial connective tissue, and there was an increase in the expression of both these epitopes at 24 mo, noteably surrounding neuron cell bodies. The expression of chondroitin-4 sulphate/dermatan sulphate was unchanged, thus implying a decreased expression of dermatan sulphate with age. Keratan sulphate and the native chondroitin sulphate epitopes were absent from the ganglia at both ages. Laminin expression was increased in the aged ganglia. It is therefore clear that the constituents of the extracellular matrix are not constant throughout the adult lifespan and that the extracellular matrix may influence neuronal survival in old age. This is the first report characterising age-related changes in the extracellular matrix of autonomic ganglia.

In Stockwell and Scott's commentary on our papers, in which we concluded from a significant mass of data (a fraction of which was presented in the paper) that we questioned the notion of ‘global patterns’ of matrix distributions within articular cartilage. In part, this was based on the distribution of keratan sulphate and chondroitin sulphate within the knee (both tibial and femoral compartments although only the latter was illustrated in the paper when referring to adult animals) of our model, the marsupial Monodelphis domestica.

The prevalence of a notch in the anterior margin of the glenoid cavity of 236 scapulae (118 female, 118 male) was investigated. The notch was found in 129 scapulae (55%) and gave rise to a pear-shaped cavity. In 107 scapulae (45%) the notch was absent, the shape of the cavity being oval. No sex difference was found in the prevalence of the notch. If a distinct notch exists, the glenoid labrum is not attached to bone at the notch and is therefore liable to be sheared off (Bankart lesion).

Double-label immunocytochemistry was used to investigate the colocalisation of various neuropeptides and the enzymes nitric oxide synthase (NOS) and tyrosine hydroxylase (TH) in intramural ganglia of the human male urinary bladder neck and trigone. Postmortem specimens were obtained from 7 male infants and children ranging in age from 2 mo to 3 y who had died as a result of cot death or accidental trauma. On average 60% of the intramural neurons were non-TH-immunoreactive (-IR) (i.e. presumptive cholinergic) and 40% were TH- and DbβH-IR (i.e. noradrenergic). Within the non-TH-IR population, calcitonin gene-related peptide (CGRP) was found in 65% of cells, neuropeptide Y (NPY) in 90%, nitric oxide synthase (NOS) in 45%, somatostatin (SOM) in 90%, and vasoactive intestinal polypeptide (VIP) in 40%. The corresponding values for the TH-IR neurons were CGRP (54%), NPY (70%), NOS (58%), SOM (73%) and VIP (40%). All the observed bombesin (BOM)-immunoreactivity was colocalised with TH while 90% of VIP and almost all the CGRP was colocalised with NPY. Less than 5% of neurons were immunoreactive for substance P (SP) or met-enkephalin (m-ENK) and some of these also contained TH. Varicose nerve fibres were seen in close proximity to some of the intramural neurons, the majority of such varicosities showing immunoreactivity to CGRP, VIP or TH. Less common were pericellular varicosities immunoreactive to NPY, SOM or SP. These results demonstrate the neurochemical heterogeneity of intramural neurons in the human bladder neck and provide indirect evidence for the complexity of the peripheral innervation of the human urinary bladder.

In their paper on opossum cartilage, Archer et al. (1996) contend that their ‘data pose questions [ctdot ] to the idea that global patterns of matrix components exist in mammalian articular cartilages’. We believe that this statement is not supported by their data. The points at issue concern the distributions of chondroitin sulphate (CS) and keratan sulphate (KS) in adult cartilage. Earlier work showed that: (1) the highest concentrations of CS are in the midzone of the noncalcified layer of the cartilage (Stockwell & Scott, 1967; Maroudas et al. 1969; Lemperg et al. 1974), while the authors' opossum data apparently show that the intensity of staining with Safranin O (and various antibodies specific for CS) ‘is greatest in the upper third of the tissue depth’; (2) in the noncalcified cartilage, the concentration of KS increases with distance from the articular surface (Stockwell & Scott, 1967; Maroudas et al. 1969; Venn, 1978). By contrast in the opossum, the authors state that there was ‘intense labelling throughout the upper half of the articular cartilage depth’. They considered that this finding, in particular, questions ‘the notions which link KS substitution with conditions of low oxygen tension’, since articular cartilage generally obtains its nutrient and O2 through the articular surface and thence by diffusion through the tissue depth.

Thirty-two human vertebral columns were selected from the Kanazawa Collection at the University of Kanazawa, Japan. The superior articular joint surface was categorised into the thoracic type and the lumbar type, and the pattern of the change from one type to the other in the lower thoracic and upper lumbar region was examined. In 21 of 32 cases (66%), the change from the thoracic to the lumbar type occurred over 2 vertebral segments, either between the 12th thoracic and 1st lumbar vertebrae (44%) or between the 11th and 12th thoracic vertebrae (22%). In the remaining 11 cases (34%), the change occurred over 3 vertebral segments, with a transitional type of articular surface. The change from the thoracic to lumbar type of articular surface has been believed to occur over 2 vertebral segments, but occurs over 3 segments in as many as 34% of the articular surfaces.

The cell population and distribution of NADPH-diaphorase positive and NOS immunoreactive intramural ganglion cells were examined on stretched whole-mount preparations of the guinea pig urinary bladder which was divided into 3 regions: base, body and dome. The results showed that the highest frequency both of NADPH-d and NOS positive neurons was observed in the bladder base. Cell counts in the whole bladder showed that the number of NADPH-d positive neurons was much more than that of NOS immunoreactive neurons. Using neuron specific enolase (NSE) positive neurons as a reference (100%), NADPH-d positive neurons accounted for 84% while NOS immunoreactive neurons only made up 45% of the total neuronal population. These results, along with previous studies on the function of nitric oxide, suggest that nitric oxide may be involved in the relaxation activity in the bladder base during micturition. The significant difference in the number of NADPH-d positive and NOS immunoreactive neurons suggests that the localisation of one enzyme does not necessarily reflect the presence of the other.

Complex arborisations occurs between the inferior ganglion of the vagus nerve and the superior cervical sympathetic ganglion (Braeucker, 1923; Fick, 1926; Siwe, 1931; Hoffman, 1957). The superior cervical sympathetic ganglion sends branches to the internal and the external carotid arteries, the inferior vagal ganglion, the superior laryngeal nerve and the cervical nerves, and provides superior cardiac and thyroid branches as well as the trunk that descends directly to the middle and inferior cervical sympathetic ganglia. There are communications between the vagus nerve (laryngeal branches) and the superior cervical sympathetic ganglion (SCSG). Cannizzaro et al. (1991) and Zerilli et al. (1994) reported abnormalities of sympathetic function among the effects of injury to the superior laryngeal nerve during thyroid surgery. The interconnections between these various nerves are therefore of clinical importance. We document here a rare example of complex communication between the vagus nerve and the SCSG in dissections of 113 adult (78 male, 35 female) Japanese cadavers. Both sides were examined in 88 cases, the right only in 18 and the left only in 7 (i.e. 201 sides in total).

We have previously shown that the neurovascular anatomy and length of the long head of biceps femoris (LHBF) is suitable for its possible use in the creation of a dynamic perianal myoplasty to restore faecal continence. If intramuscular arterioarterial anastomoses exist between a muscle's vascular pedicles then the delay procedure, i.e. ligating the vascular pedicles to the transposed section of the muscle, 4–6 wk before transposition, can be used to improve blood flow to the distal part of the transposed muscle. The intramuscular arterial anatomy of 20 biceps femoris muscles was shown by dissection (14) or radiographically (6). The mean entry point of the upper major vascular pedicle to the LHBF was 12 cm (s.d. 3.3) and the mean length of the LHBF was 36.8 cm (s.d. 1.8). Therefore the ratio between upper major vascular pedicle entry point and muscle length in 95% of specimens was 0.33 (0.2). In the present study intramuscular arterioarterial anastomoses were found between the arterial pedicles to the non-transposed and transposed sections of the LHBF and between the long and short heads of this muscle. Using the delay procedure would therefore theoretically allow the intramuscular arterioarterial anastomoses between the arterial pedicle to the nontransposed and transposed sections of the LHBF to open up enhancing blood flow to the distal part of the myoplasty.

I am writing to the Journal of Anatomy as a component of the response by Archer et al. to a letter to the Editor submitted by Professors Robin Stockwell and John Scott. Professor Archer has asked me to comment on their claims and some matters regarding antibodies used in the studies in question. My responses to these issues are indicated below.

Anatomical Society of Great Britain and Ireland

15–17 July 1997. Medical School, University of Edinburgh.

6–8 January 1998. University of Newcastle upon Tyne.

24–26 July 2000. University of Cambridge.

Anatomical Society Research Studentchips

Grant funding is available for Research Studentships in the anatomical sciences, commencing October 1997. Project applications are invited from members of academic staff in departments of anatomical sciences in the UK and Ireland.